Venetoclax, an orally bioavailable BCL-2 inhibitor, has been regarded as a breakthrough for the treatment of leukemia but has a wide interindividual variability and drug-drug interaction in pharmacokinetics. In this study, a simple and sensitive ultra-high performance liquid chromatography-tandem with mass spectrometry method was established and fully validated to quantify venetoclax and voriconazole simultaneously in human plasma. After protein precipitation, the analytes were separated on a Hypersil GOLD C18 column (3 µm, 2.1 × 50 mm) by gradient elution. The mass detection was operated under multiple reaction monitoring mode at m/z 868.5 →636.2 for venetoclax, 350.0 → 127.0 for voriconazole and 353.0 → 127.0 for voriconazole-D3(Internal Standard). The calibration ranges were 0.1-10 μg/mL for venetoclax and 0.05-10 μg/mL for voriconazole with correlation coefficients (r²)＞0.998. The validated method was successfully applied to the pharmacokinetic study in acute myeloid leukemia patients receiving venetoclax with or without voriconazole. The results suggested that co-treatment with 200 mg q12h voriconazole, the peak concentration of venetoclax (100 mg qd) could be raised to the same level as the 400 mg qd group. However, the trough concentration of venetoclax (100 mg qd) was much higher than that of the 400 mg qd group. Therapeutic drug monitoring might give some guidance for the adjustment of dosing regimens to guarantee the drug efficacy and safety of patients to some extent.